Comparison of TPM and original counts in DE analysis with the DESeq2 package
Raw data
- Study aim: Reveal neurological complications in patients with COVID-19
- Study summary: Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids were infected with SARS-CoV-2 for 72hrs and analyzed with bulk RNA-seq.
- Samples: Mock- or SARS-CoV-2-infected neuronal cells
Loading packages
library(data.table)
library(rmarkdown)
library(AnnotationHub)
library(tidyverse)
library(tximport)
library(ggplot2)
library(DESeq2)
library(pheatmap)
library(gridExtra)
library(ggplotify)
library(ggrepel)
library(UpSetR)Setting AnnotationHub
AnnotationSpecies <- "Homo sapiens" # Assign your species
ah <- AnnotationHub(hub=getAnnotationHubOption("URL")) # Bring annotation DBRunning AnnotationHub
ahQuery <- query(ah, c("OrgDb", AnnotationSpecies)) # Filter annotation of interest
if (length(ahQuery) == 1) {
DBName <- names(ahQuery)
} else if (length(ahQuery) > 1) {
DBName <- names(ahQuery)[1]
} else {
print("You don't have a valid DB")
rmarkdown::render()
}
AnnoDb <- ah[[DBName]] # Store into an OrgDb object
# Explore your OrgDb object with following accessors:
# columns(AnnpDb)
# keytypes(AnnoDb)
# keys(AnnoDb, keytype=..)
# select(AnnoDb, keys=.., columns=.., keytype=...)
AnnoKey <- keys(AnnoDb, keytype="ENSEMBLTRANS")
# Note: Annotation has to be done with not genome but transcripts
AnnoDb <- select(AnnoDb,
AnnoKey,
keytype="ENSEMBLTRANS",
columns="SYMBOL")Checking out the AnnotationHub output
# Check if your AnnoDb has been extracted and saved correctely
class(AnnoDb)## [1] "data.frame"head(AnnoDb)## ENSEMBLTRANS SYMBOL
## 1 ENST00000543404 A2MP1
## 2 ENST00000566278 A2MP1
## 3 ENST00000545343 A2MP1
## 4 ENST00000544183 A2MP1
## 5 ENST00000286479 NAT2
## 6 ENST00000520116 NAT2Defining file name and path for .sf files
.sf files have been created from fastq data by salmon
# This code chunk needs to be written by yourself
# Define sample names
SampleNames <- c("Mock_72hpi_S1",
"Mock_72hpi_S2",
"Mock_72hpi_S3",
"SARS-CoV-2_72hpi_S7",
"SARS-CoV-2_72hpi_S8",
"SARS-CoV-2_72hpi_S9")
# Define group level
GroupLevel <- c("Mock", "COVID")
# Define contrast for DE analysis
Contrast <- c("Group", "COVID", "Mock")
# Define a vector for comparing TPM vs Counts effect
TvC <- c("TPM", "Counts")
# Define .sf file path
sf <- c(paste0(SampleNames,
".fastq.gz.salmon_quant/quant.sf"))
# Define sample groups
group <- c(rep("Mock", 3), rep("COVID", 3))
# Create metadata
metadata <- data.frame(Sample=factor(SampleNames, levels=SampleNames),
Group=factor(group, levels=GroupLevel),
Path=sf)
rownames(metadata) <- SampleNames
# Explore the metadata
print(metadata)## Sample Group
## Mock_72hpi_S1 Mock_72hpi_S1 Mock
## Mock_72hpi_S2 Mock_72hpi_S2 Mock
## Mock_72hpi_S3 Mock_72hpi_S3 Mock
## SARS-CoV-2_72hpi_S7 SARS-CoV-2_72hpi_S7 COVID
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S8 COVID
## SARS-CoV-2_72hpi_S9 SARS-CoV-2_72hpi_S9 COVID
## Path
## Mock_72hpi_S1 Mock_72hpi_S1.fastq.gz.salmon_quant/quant.sf
## Mock_72hpi_S2 Mock_72hpi_S2.fastq.gz.salmon_quant/quant.sf
## Mock_72hpi_S3 Mock_72hpi_S3.fastq.gz.salmon_quant/quant.sf
## SARS-CoV-2_72hpi_S7 SARS-CoV-2_72hpi_S7.fastq.gz.salmon_quant/quant.sf
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S8.fastq.gz.salmon_quant/quant.sf
## SARS-CoV-2_72hpi_S9 SARS-CoV-2_72hpi_S9.fastq.gz.salmon_quant/quant.sfConverting .sf files to txi list
- txi_tpm: stores TPM with the argument “countsFromAbundance=”lengthScaledTPM"
- txi_counts: stores original counts
- Despite that the TPM matrix is not recommended as an input for DE analysis due to the fact that it doesn’t account gene length, TPM stored in a tximport (by tximport(…, countsFromAbundance=“lengthScaledTPM”)) can be used for DE analysis by being inputted with the DESeqDataSetFromTximport() funtion in DESeq2 workflow as the gene length is automatically adjusted by DESeqDataSetFromTximport().
- In this project, two txi objects were created with or without the countsFromAbundance=“lengthScaledTPM” argument and compared in downstream DE analysis.
- If you don’t want gene-level summarization, set txOut=TRUE.
- References: tximport doc, DESeq2 doc “Why unnormalized counts?”, Soneson et al. 2016, Developer Dr. Love’s comment, Harvard Chan Bioinformatics Core workshop
# Assign sample names to the input (.sf) file path
names(sf) <- SampleNames
# Run tximport
# tpm vs original counts
# input sf: a factor of all .sf files' path
txi_tpm <- tximport(sf,
type="salmon",
tx2gene=AnnoDb,
countsFromAbundance="lengthScaledTPM", # Extracts TPM
ignoreTxVersion=T)
txi_counts <- tximport(sf,
type="salmon",
tx2gene=AnnoDb,
ignoreTxVersion=T) Exploring the txi outputs
# tpm
head(txi_tpm$counts)## Mock_72hpi_S1 Mock_72hpi_S2 Mock_72hpi_S3 SARS-CoV-2_72hpi_S7
## A2MP1 5.705442 5.571614 0.8060672 0.00000
## A3GALT2 0.000000 0.000000 0.0000000 0.00000
## A4GNT 0.000000 2.953081 0.9964319 0.00000
## AACSP1 14.635652 19.646913 9.0911703 19.54913
## AADACL2 0.000000 0.000000 0.0000000 0.00000
## AADACL4 1.001855 0.000000 1.9900495 0.00000
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S9
## A2MP1 0.000000 7.265574
## A3GALT2 0.000000 0.000000
## A4GNT 0.000000 1.009116
## AACSP1 18.772579 4.560492
## AADACL2 0.000000 0.000000
## AADACL4 2.009001 0.000000dim(txi_tpm$counts)## [1] 11013 6# counts
head(txi_counts$counts)## Mock_72hpi_S1 Mock_72hpi_S2 Mock_72hpi_S3 SARS-CoV-2_72hpi_S7
## A2MP1 7 7 1 0
## A3GALT2 0 0 0 0
## A4GNT 0 3 1 0
## AACSP1 16 22 10 21
## AADACL2 0 0 0 0
## AADACL4 1 0 2 0
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S9
## A2MP1 0 3
## A3GALT2 0 0
## A4GNT 0 1
## AACSP1 10 5
## AADACL2 0 0
## AADACL4 2 0dim(txi_counts$counts)## [1] 11013 6Creating DESeq objects from txi and VST
- Note: The tximport-to-DESeq2 approach uses estimated gene counts from the transcript abundance quantifiers, but not normalized counts.
- The DESeqDataSetFromTximport() function generated an DESeq object (aka dds) with the txi input.
- vst() was run to perform variance stabilizing transformation instead of rlog() which takes longer time with similar characteristics.
- The vsd object created by vst() is used for not DE analysis but QC.
- References: DESeq2 doc “Transcript abundance files”, DESeq2 doc “Variance stabilizing transformation”
# Set a function creating dds and vsd
dds_vsd_fn <- function(txi) {
# Create a DESeq object (so-calledd dds)
des <- DESeqDataSetFromTximport(txi,
colData=metadata,
design=~Group)
# Create a vsd object (so-called vsd)
ves <- vst(des, blind=T)
# Output them as a list
return(list(dds=des, vsd=ves))
}
TPM <- dds_vsd_fn(txi_tpm)
Counts <- dds_vsd_fn(txi_counts)
# Outputs
# dds: TPM/Counts[[1]] or TPM/Counts[['dds']]
# vsd: TPM/Counts[[2]] or TPM/Counts[['vsd']]Exploring created dds
# TPM
TPM[[1]]## class: DESeqDataSet
## dim: 11013 6
## metadata(1): version
## assays(1): counts
## rownames(11013): A2MP1 A3GALT2 ... ZXDA ZXDB
## rowData names(0):
## colnames(6): Mock_72hpi_S1 Mock_72hpi_S2 ... SARS-CoV-2_72hpi_S8
## SARS-CoV-2_72hpi_S9
## colData names(3): Sample Group Pathhead(counts(TPM[[1]]))## Mock_72hpi_S1 Mock_72hpi_S2 Mock_72hpi_S3 SARS-CoV-2_72hpi_S7
## A2MP1 6 6 1 0
## A3GALT2 0 0 0 0
## A4GNT 0 3 1 0
## AACSP1 15 20 9 20
## AADACL2 0 0 0 0
## AADACL4 1 0 2 0
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S9
## A2MP1 0 7
## A3GALT2 0 0
## A4GNT 0 1
## AACSP1 19 5
## AADACL2 0 0
## AADACL4 2 0# Counts
Counts[[1]]## class: DESeqDataSet
## dim: 11013 6
## metadata(1): version
## assays(2): counts avgTxLength
## rownames(11013): A2MP1 A3GALT2 ... ZXDA ZXDB
## rowData names(0):
## colnames(6): Mock_72hpi_S1 Mock_72hpi_S2 ... SARS-CoV-2_72hpi_S8
## SARS-CoV-2_72hpi_S9
## colData names(3): Sample Group Pathhead(counts(Counts[[1]]))## Mock_72hpi_S1 Mock_72hpi_S2 Mock_72hpi_S3 SARS-CoV-2_72hpi_S7
## A2MP1 7 7 1 0
## A3GALT2 0 0 0 0
## A4GNT 0 3 1 0
## AACSP1 16 22 10 21
## AADACL2 0 0 0 0
## AADACL4 1 0 2 0
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S9
## A2MP1 0 3
## A3GALT2 0 0
## A4GNT 0 1
## AACSP1 10 5
## AADACL2 0 0
## AADACL4 2 0Exploring created vsd
# TPM
TPM[[2]]## class: DESeqTransform
## dim: 11013 6
## metadata(1): version
## assays(1): ''
## rownames(11013): A2MP1 A3GALT2 ... ZXDA ZXDB
## rowData names(4): baseMean baseVar allZero dispFit
## colnames(6): Mock_72hpi_S1 Mock_72hpi_S2 ... SARS-CoV-2_72hpi_S8
## SARS-CoV-2_72hpi_S9
## colData names(4): Sample Group Path sizeFactor# Counts
Counts[[2]]## class: DESeqTransform
## dim: 11013 6
## metadata(1): version
## assays(1): ''
## rownames(11013): A2MP1 A3GALT2 ... ZXDA ZXDB
## rowData names(4): baseMean baseVar allZero dispFit
## colnames(6): Mock_72hpi_S1 Mock_72hpi_S2 ... SARS-CoV-2_72hpi_S8
## SARS-CoV-2_72hpi_S9
## colData names(3): Sample Group PathEstimating size factors, dispersions, and conducting Wald Test
- Dispersion is calculated as a measure of variation instead of variance since variance gets larger when gene expression gets higher.
- Wald test is the default setting of DESeq2 which tests null hypothesis between two groups. You should use Likelihood ratio test (LRT) when comparing more than two groups.
- Messages when “Counts <- DESeqPrep_fn(Counts)” was run:
using ‘avgTxLength’ from assays(dds), correcting for library size gene-wise dispersion estimates mean-dispersion relationship final dispersion estimates
- References: Harvard Chan Bioinformatics Core workshop I, Harvard Chan Bioinformatics Core workshop II, Harvard Chan Bioinformatics Core workshop III, DESeq2 "Wald test indivisual steps, DESeq2 doc “Likelihood ratio test”
# Set a function estimating size factors, dispersions, and perform wald test
DESeqPrep_fn <- function(List) {
List[[1]] <- estimateSizeFactors(List[[1]])
List[[1]] <- estimateDispersions(List[[1]])
List[[1]] <- nbinomWaldTest(List[[1]])
return(List)
}
# Update dds with the function
Counts <- DESeqPrep_fn(Counts)
TPM <- DESeqPrep_fn(TPM)Exploring size factors
sizeFactors(Counts[[1]])## NULLsizeFactors(TPM[[1]])## Mock_72hpi_S1 Mock_72hpi_S2 Mock_72hpi_S3 SARS-CoV-2_72hpi_S7
## 1.0991742 1.0489678 0.8749073 1.3751270
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S9
## 0.7673324 1.0049385# Size factors don't exist in the Counts dds!
# Normalization factors are calculated in the Counts dds instead!
assays(Counts[[1]])## List of length 6
## names(6): counts avgTxLength normalizationFactors mu H cooksassays(TPM[[1]])## List of length 4
## names(4): counts mu H cookscolData(Counts[[1]])## DataFrame with 6 rows and 3 columns
## Sample Group Path
## <factor> <factor> <character>
## Mock_72hpi_S1 Mock_72hpi_S1 Mock Mock_72hpi_S1.fastq...
## Mock_72hpi_S2 Mock_72hpi_S2 Mock Mock_72hpi_S2.fastq...
## Mock_72hpi_S3 Mock_72hpi_S3 Mock Mock_72hpi_S3.fastq...
## SARS-CoV-2_72hpi_S7 SARS-CoV-2_72hpi_S7 COVID SARS-CoV-2_72hpi_S7...
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S8 COVID SARS-CoV-2_72hpi_S8...
## SARS-CoV-2_72hpi_S9 SARS-CoV-2_72hpi_S9 COVID SARS-CoV-2_72hpi_S9...colData(TPM[[1]])## DataFrame with 6 rows and 4 columns
## Sample Group Path
## <factor> <factor> <character>
## Mock_72hpi_S1 Mock_72hpi_S1 Mock Mock_72hpi_S1.fastq...
## Mock_72hpi_S2 Mock_72hpi_S2 Mock Mock_72hpi_S2.fastq...
## Mock_72hpi_S3 Mock_72hpi_S3 Mock Mock_72hpi_S3.fastq...
## SARS-CoV-2_72hpi_S7 SARS-CoV-2_72hpi_S7 COVID SARS-CoV-2_72hpi_S7...
## SARS-CoV-2_72hpi_S8 SARS-CoV-2_72hpi_S8 COVID SARS-CoV-2_72hpi_S8...
## SARS-CoV-2_72hpi_S9 SARS-CoV-2_72hpi_S9 COVID SARS-CoV-2_72hpi_S9...
## sizeFactor
## <numeric>
## Mock_72hpi_S1 1.099174
## Mock_72hpi_S2 1.048968
## Mock_72hpi_S3 0.874907
## SARS-CoV-2_72hpi_S7 1.375127
## SARS-CoV-2_72hpi_S8 0.767332
## SARS-CoV-2_72hpi_S9 1.004939Plotting the size factors in TPM
- The size factors are only available from TPM dds
- Blue dashed line: normalization factor = 1
# Extract and save the size factors in a data frame
sizeFactor <- as.data.frame(round(sizeFactors(TPM[[1]]), 3))
colnames(sizeFactor) <- 'Size_Factor'
sizeFactor <- sizeFactor %>%
rownames_to_column(var="Sample") %>%
inner_join(metadata[, 1:ncol(metadata)-1], by="Sample")
# Create a plot comparing the size factors by sample
ggplot(sizeFactor, aes(x=Sample,
y=Size_Factor,
fill=Group,
label=Size_Factor)) +
geom_bar(stat="identity", width=0.8) +
theme_bw() +
ggtitle("Size Factors in TPM-DESeq") +
geom_text(vjust=1.5) +
theme(axis.text.x=element_text(angle=45,
vjust=0.5)) + ylab("Size Factor") + geom_hline(yintercept=1, color="blue", linetype="dashed")
Plotting nornalization factors in the Counts
- DESeq2 performs an internal normalization where geometric mean is calculated for each gene across all samples. The counts for a gene in each sample is then divided by this mean. The median of these ratios in a sample is the size factor for that sample.
- Blue dashed line: normalization factor = 1
- Colored dots: normlization factors per gene (y-axis) in each sample
- Box plots: distribution of the normalization facters in each group (see the second plot)
- Reference: DESeq2 doc “Sample-/gene-dependent normalization factors”
# Extract and normalization factors in a data frame
normf <- as.data.frame(normalizationFactors(Counts[[1]])) %>%
gather(Sample, Normalization_Factor) %>%
inner_join(metadata[, 1:2], by="Sample")
normf$Sample <- factor(normf$Sample, levels=SampleNames)
normf$Group <- factor(normf$Group, levels=GroupLevel)
# Create a scatter plot showing distribution of normalization factors
normFactors_plot <- ggplot(normf,
aes(x=Sample, y=Normalization_Factor)) +
geom_jitter(alpha=0.5, aes(color=Group)) +
# Add a boxplot to provide statistics in each sample
geom_boxplot(aes(x=Sample, y=Normalization_Factor),
outlier.shape=NA, alpha=0.5) +
theme_bw() +
ggtitle("Normalization Factors in Counts-DESeq") +
theme(axis.text.x=element_text(angle=45,
vjust=0.5)) +
ylab("Normalization Factor / Gene") +
# Add a dashed horizontal line to indicate where normalization factor=1
geom_hline(yintercept=1,
color="blue",
linetype="dashed")
# Print the normalization factor scatter plot
print(normFactors_plot)
# Print the same plot with larger y-magnification in order to observe the box plot
normFactors_plot +
ylim(0.5, 1.5)
Results & Discussion 1:
- Patterns of the size factors (from TPM) and the normalization factors per gene (from Counts) by sample were similar in terms of magnitude
Setting functions for QC plots
- Reference: DESeq2 doc “Principal component plot of the samples”, DESeq2 doc “Heatmap of the sample-to-sample distances”
# Assigne what to compare
GroupOfInterest <- Contrast[1]
# Set a function for a PCA plot
QCPCA_fn <- function(inputList, Title) {
plotPCA(inputList[[2]], # takes vsd
intgroup=GroupOfInterest) + theme_bw() + ggtitle(Title)
}
# Set heatmap annotation
ColOfInterest <- !colnames(metadata) %in% c("Sample", "Path")
HeatmapAnno <- as.data.frame(metadata[, ColOfInterest])
rownames(HeatmapAnno) <- SampleNames
colnames(HeatmapAnno) <- colnames(metadata)[ColOfInterest]
# Set a function for a correlation heatmap
QCcorrHeatmap_fn <- function(inputList, Title) {
# Extract transformed count matrix
mtx <- assay(inputList[[2]]) # takes vsd
# Calculate correlation and store in the matrix
mtx <- cor(mtx)
# Create a correlation heatmap
return(pheatmap(mtx,
annotation=HeatmapAnno,
main=paste("Sample Correlation Heatmap:",
Title)))
}Sample QC: Principal Component Analysis
- Checkpoints in PCA: source of variation, sample outlier
grid.arrange(QCPCA_fn(TPM, "QC PCA: TPM"),
QCPCA_fn(Counts, "QC PCA: Counts"),
nrow=2)
Sample QC: Sample Correlation Heatmap
- Checkpoints of correlation heatmap: distance between samples, correlation in a group
- Upper: TPM input
- Lower: Counts input
# TPM
QCcorrHeatmap_fn(TPM, "TPM") 
# Counts
QCcorrHeatmap_fn(Counts, "Counts") 
Results & Discussion 2:
- Unsupervised learning-mediated sample QC resulted in highly similar patterns between TPM and Counts
Running DE analysis
# Create a list for TPM and Counts dds
ddsList <- list(TPM=TPM[[1]], Counts=Counts[[1]])
for (x in TvC) {
# Run DESeq()
ddsList[[x]] <- DESeq(ddsList[[x]])
print(resultsNames(ddsList[[x]]))
}## [1] "Intercept" "Group_COVID_vs_Mock"
## [1] "Intercept" "Group_COVID_vs_Mock"Creating dispersion plots
- Dispersion is important since estimation by DESeq2 algorithm is based on the assumption that genes with similar expression levels have similar dispersion. If an RNA-seq dataset doesn’t satisfy this assumption, use other DE algorithms than DESeq2.
- References: DESeq2 doc "Dispersion plot and fitting alternatives, Harvard Chan Bioinformatics Core workshop
# Plot dispersion
for (x in TvC) {
plotDispEsts(ddsList[[x]],
ylab="Dispersion",
xlab="Mean of Normalized Counts",
main=paste("Dispersion of", x, "Input"))
}
Results & Discussion 3:
- Both inputs gave a goot fit
- No major difference was seen in the dispersion plots between two conditions
Shrinking effect size
- Shrinkage reduces false positives
- Magnitude of shrinkage is affected by dispersion and sample size
- When the argument type is set to “apeglm”, the coef argument is used instead of constrast. In this dataset, you can set coef=Coef where Coef <- resultsNames(ddsList[[1]]).
- When the type is set to “normal”, the argument contrast is set as shown below.
- References: DESeq2 doc “Alternative shrinkage estimators”, Harvard Chan Bioinformatics Core workshop
# Create an empty list for shrunken dds
shr_ddsList <- list(TPM=c(), Counts=c())
for (x in TvC) {
# shrink
shr_ddsList[[x]] <- lfcShrink(ddsList[[x]],
contrast=Contrast, # contrast
type="normal") # is paired with "normal" type
}Extracting log2FoldChange and p-values with or without shrinkage
- The alpha denotes threshold of false discovery rate (FDR) assigned by users.
- In this analysis, the alpha is set to 0.1
# Set FDR threshold
alpha=0.1
# FDR threshold vector
FDRv=c("< 0.1", "> 0.1")
# Set a function cleaning table
Sig_fn <- function(df, Input) {
df <- df %>%
rownames_to_column(var="Gene") %>%
mutate(FDR=ifelse(padj < 0.1 & !is.na(padj),
FDRv[1],
FDRv[2]),
Input=Input)
return(df)
}
# Initialize lists of result tables with (resList) or without (shr_resList) shrinkage
resList <- ddsList
shr_resList <- ddsList
for (x in TvC) {
# Extract results
resList[[x]] <- as.data.frame(results(ddsList[[x]],
contrast=Contrast,
alpha=alpha))
shr_resList[[x]] <- as.data.frame(shr_ddsList[[x]])
# clean the data frame
resList[[x]] <- Sig_fn(resList[[x]], x)
shr_resList[[x]] <- Sig_fn(shr_resList[[x]], x)
}Exploratory data analysis of the extracted log2FoldChange tables
# No shrinkage summary
summary(resList)## Length Class Mode
## TPM 9 data.frame list
## Counts 9 data.frame listhead(resList[['TPM']])## Gene baseMean log2FoldChange lfcSE stat pvalue padj
## 1 A2MP1 3.2145217 -0.83018949 1.7557571 -0.47283846 0.6363284 NA
## 2 A3GALT2 0.0000000 NA NA NA NA NA
## 3 A4GNT 0.8330031 -1.92416012 3.0773846 -0.62525826 0.5318016 NA
## 4 AACSP1 14.5467370 0.02184858 0.7549595 0.02894006 0.9769124 0.9923554
## 5 AADACL2 0.0000000 NA NA NA NA NA
## 6 AADACL4 0.9670271 -0.35981582 2.9306060 -0.12277864 0.9022824 NA
## FDR Input
## 1 > 0.1 TPM
## 2 > 0.1 TPM
## 3 > 0.1 TPM
## 4 > 0.1 TPM
## 5 > 0.1 TPM
## 6 > 0.1 TPMhead(resList[['Counts']])## Gene baseMean log2FoldChange lfcSE stat pvalue padj
## 1 A2MP1 2.9604793 -0.90913229 1.813261 -0.50137976 0.6161039 NA
## 2 A3GALT2 0.0000000 NA NA NA NA NA
## 3 A4GNT 0.8386688 -1.90512920 3.007858 -0.63338401 0.5264829 NA
## 4 AACSP1 14.0360149 -0.06225852 0.760773 -0.08183586 0.9347772 0.9796733
## 5 AADACL2 0.0000000 NA NA NA NA NA
## 6 AADACL4 0.9793046 -0.35701152 2.864781 -0.12462089 0.9008237 NA
## FDR Input
## 1 > 0.1 Counts
## 2 > 0.1 Counts
## 3 > 0.1 Counts
## 4 > 0.1 Counts
## 5 > 0.1 Counts
## 6 > 0.1 Counts# Shrinkage summary
summary(shr_resList)## Length Class Mode
## TPM 9 data.frame list
## Counts 9 data.frame listhead(shr_resList[['TPM']])## Gene baseMean log2FoldChange lfcSE stat pvalue padj
## 1 A2MP1 3.2145217 -0.1588907 0.3391943 -0.47283846 0.6363284 NA
## 2 A3GALT2 0.0000000 NA NA NA NA NA
## 3 A4GNT 0.8330031 -0.1303038 0.2238159 -0.62525826 0.5318016 NA
## 4 AACSP1 14.5467370 0.0122880 0.4253130 0.02894006 0.9769124 0.9923554
## 5 AADACL2 0.0000000 NA NA NA NA NA
## 6 AADACL4 0.9670271 -0.0275875 0.2314025 -0.12277864 0.9022824 NA
## FDR Input
## 1 > 0.1 TPM
## 2 > 0.1 TPM
## 3 > 0.1 TPM
## 4 > 0.1 TPM
## 5 > 0.1 TPM
## 6 > 0.1 TPMhead(shr_resList[['Counts']])## Gene baseMean log2FoldChange lfcSE stat pvalue padj
## 1 A2MP1 2.9604793 -0.15661253 0.3347863 -0.50137976 0.6161039 NA
## 2 A3GALT2 0.0000000 NA NA NA NA NA
## 3 A4GNT 0.8386688 -0.13471856 0.2283621 -0.63338401 0.5264829 NA
## 4 AACSP1 14.0360149 -0.03454845 0.4258173 -0.08183586 0.9347772 0.9796733
## 5 AADACL2 0.0000000 NA NA NA NA NA
## 6 AADACL4 0.9793046 -0.02852632 0.2359889 -0.12462089 0.9008237 NA
## FDR Input
## 1 > 0.1 Counts
## 2 > 0.1 Counts
## 3 > 0.1 Counts
## 4 > 0.1 Counts
## 5 > 0.1 Counts
## 6 > 0.1 CountsExploring mean-difference relationship with MA plots
- Reference: DESeq2 doc “MA-plot”
# Set ylim: has to adjusted by users depending on data
yl <- c(-20, 20)
# Set min log2 fold change of interest
mLog <- c(-1, 1)
# Define a function creating an MA plot
MA_fn <- function(List, Shr) {
MAList <- ddsList
for (i in 1:2) {
MAplot <- ggplot(List[[i]],
aes(x=baseMean,
y=log2FoldChange,
color=FDR)) + geom_point() + scale_x_log10() + theme_bw() + scale_color_manual(values=c("blue", "grey")) + ggtitle(paste("MA plot:", names(List)[i], "Input with", Shr)) + ylim(yl[1], yl[2])+ geom_hline(yintercept=c(mLog[1], mLog[2]), linetype="dashed", color="red")
MAList[[i]] <- MAplot
}
return(MAList)
}
# Create MA plots with or without shrinkage and store in a list
MA <- MA_fn(resList, "No Shrinkage")
shr_MA <- MA_fn(shr_resList, "Shrinkage")Displaying MA plots
- x-axis: expression level (baseMean))
- y-axis: fold change (log2FoldChange)
- Red dashed lines: log2FoldChange = -1 and 1
- Upper: TPM with (right) or without (left) shrinkage
- Lower: Counts with (right) or without (left) shrinkage
# TPM with or without shrinkage
grid.arrange(MA[[1]], shr_MA[[1]], nrow=1)
# TPM with or without shrinkage
grid.arrange(MA[[2]], shr_MA[[2]], nrow=1)
Results & Discussion 4:
- Both inputs gave fairly identical MA plots
Exploring distribution of false discovery rate (FDR)
- Distribution of adjusted p-val (FDR) was presented
- x-axis: FDR
- y-axis: Number of genes
- Black dashed line: FDR = 0.1
# Combining total data table
res <- rbind(shr_resList[['TPM']], shr_resList[['Counts']])
res$Input <- factor(res$Input, levels=TvC) # TvC=c("TPM", "Counts")
# Create a plot presenting distribution of FDR
ggplot(res,
aes(x=padj, color=Input)) +
geom_density(size=1, aes(y=..count..)) +
theme_bw() +
ggtitle("Distribution of False Discovery Rate (FDR)") +
xlab("Adjusted P-Value (FDR)") +
ylab("Count") +
geom_vline(xintercept=alpha,
color="black",
linetype="dashed",
size=1) +
scale_x_continuous(breaks=seq(0, 1, by=0.1)) 
Results & Discussion 5:
- Both inputs showed highly similar FDR distribution patterns across the FDR range.
- When the FDR > 0.7, more genes were counted in the TPM input than in the Counts.
Volcano plots
- Black dashed lines: log2FoldChange = -1 and 1
- x-axis: gene expression level (log2FoldChange)
- y-axis: number of genes
# Set xlim for volcano plots
xlim=c(-6, 6) # has to be assined by users
# Set a basic volcano plot function
Volcano_fn <- function(df, Label=NULL) {
ggplot(df,
aes(x=log2FoldChange,
y= -log10(padj),
color=FDR,
label=Label)) +
geom_point() +
facet_grid(.~Input) +
theme_bw() +
scale_color_manual(values=c("blue", "grey")) +
ggtitle("Volcano Plot") +
ylab("-log10(padj)") +
theme(strip.text.x=element_text(size=12)) +
geom_vline(xintercept=c(mLog[1], mLog[2]),
color="red",
linetype="dashed",
size=1) +
xlim(xlim[1], xlim[2])
}
# Display the volcano plots by input
Volcano_fn(res)
Volcano plots with promising genes
- Log odds threshold (y-axis): > 20
# Assign log odds threshold
LogOddsCut=20
# Add a column indicating high log odds genes
res <- res %>%
mutate(Label=ifelse(-log10(padj) > LogOddsCut,
Gene,
""))
# Display the genes with volcano plots
Volcano_fn(res, Label=res$Label) + geom_text_repel(color="black")
Results & Discussion 6:
- Both inputs gave nearly identical volcano plots
Exploring distribution of log2FoldChange by input type
- Black dashed lines: log2FoldChange = -1 and 1
- x-axis: gene expression level (log2FoldChange)
- y-axis: number of genes
ggplot(res[res$FDR == "< 0.1", ], # Subset rows with FDR < alpha
aes(x=log2FoldChange,
color=Input)) +
geom_density(size=1, aes(y=..count..)) +
theme_bw() +
ylab("Count") +
geom_vline(xintercept=c(mLog[1], mLog[2]),
color="black",
linetype="dashed", size=1) +
ggtitle("Distribution of Log2 Folds by Input Type") +
xlim(xlim[1], xlim[2])
Results & Discussion 7:
- Both inputs showed nearly identical fold change distribution
Exploring expression profiling with normalized count data
- Normalized count matrices are extracted from dds objects and filtered with thresholds set at FDR and log2FoldChange
- The heatmaps display z-scores of the normalized counts
- lowfdrList: a list of matrices filtered by FDR < alpha
- highfoldList: a list of matrices filtered by FDR < alpha AND absolute log2FoldChange > user’s minimum threshold (mLog)
- In this analysis, mLog = 1
- References: Harvard Chan Bioinformatics Core workshop, DESeq2 doc “Heatmap of the count matrix”
# Initialize a list
lowfdrList <- ddsList # A list for normalized counts matrix with FDR below alpha
highfoldList <- ddsList # A list for normalized counts with log2foldchange over minLog
for (x in TvC) {
# Create filtering vectors with alpha and log2foldchange
BelowAlpha <- shr_resList[[x]]$FDR == FDRv[1]
overmLog <- abs(shr_resList[[x]]$log2FoldChange) > mLog[2] # mLog has been set to c(-1, 1) previously
# Extract transformed counts from vsd objects (TPM[['vsd']] and Counts[['vsd']])
if (x == "TPM") {
normCounts <- counts(TPM[['dds']], normalized=T)
} else {
normCounts <- counts(Counts[['dds']], normalized=T)
}
# Update the normalized count matrix with FDR below alpha
lowfdrList[[x]] <- normCounts[BelowAlpha, ]
highfoldList[[x]] <- normCounts[BelowAlpha & overmLog, ]
summary(lowfdrList[[x]])
summary(highfoldList[[x]])
}
# Initialize map lists
lowfdrMap <- ddsList
highfoldMap <- ddsList
# Set a function creating a heatmap
ProfileHeatmap_fn <- function(inputmatrix, Title1, Title2, Title3=NULL) {
as.ggplot(pheatmap(inputmatrix,
annotation=HeatmapAnno,
scale="row", # presents z-score instead of counts
show_rownames=F,
main=paste("Transcription Profiles with",
Title1,
"input and",
Title2,
alpha,
Title3)))
}
# Create and save heatmaps
for (x in TvC) {
lowfdrMap[[x]] <- ProfileHeatmap_fn(lowfdrList[[x]],
Title1=x,
Title2="FDR <")
highfoldMap[[x]] <- ProfileHeatmap_fn(highfoldList[[x]],
Title1=x,
Title2="FDR <",
Title3=paste("+ Absolte Log2 Fold Change >", mLog[2]))
}
Results & Discussion 8:
- No major difference is seen bewteen the TPM and Counts
NA statistics: zero count genes & outlier genes
When NAs appear in
- log2FoldChange: zero counts in all samples
- padj: too little information
- pval & padj: at least one replicate was an outlier
# Count number of NA genes
type=c("Zero Counts", "Outliers", "Total NA Genes")
NAstat <- res %>%
group_by(Input) %>%
summarize(zero=sum(is.na(log2FoldChange)),
outlier=sum(is.na(pvalue) & is.na(padj))) %>%
mutate(total=sum(zero, outlier)) %>%
gather(Type, Number, -Input) %>%
mutate(Type=factor(case_when(Type == "zero" ~ type[1],
Type == "outlier" ~ type[2],
Type == "total" ~ type[3]),
levels=type))
# Plot number of NA genes
ggplot(NAstat, aes(x=Type, y=Number, group=Input, fill=Input, label=Number)) +
geom_bar(stat="identity", position="dodge") +
theme_bw() +
geom_text(position=position_dodge(width=1), vjust=1.5) +
ggtitle("Number of NA Genes") +
ylab("Number of Genes")
Results & Discussion 9:
- Both inputs produced no major difference in the number of NA genes
Ranking DEGs with the TPM and original count inputs
- FDRrankList: ranked by FDR
- lfcList: ranked by absolute fold change
- UPlfcList: ranked by magnitude of fold change increase
- DOWNlfcList: ranked by manitude of fold change decrease
# Create a new list having DE table with FDR below alpha
lowfdr_resList <- shr_resList
for (x in TvC) {
lowfdr_resList[[x]] <- filter(shr_resList[[x]],
FDR == FDRv[1]) %>%
as.data.table()
}
# Initialize new lists in order to store rank-updated result DE tables
FDRrankList <- lowfdr_resList
lfcList <- lowfdr_resList
UPlfcList <- lowfdr_resList
DOWNlfcList <- lowfdr_resList
# Set a function creating a column for the rank
Ranking_fn <- function(x) {mutate(x, Rank=1:nrow(x))}
for (x in TvC) {
# Rearrange genes with FDR
FDRrankList[[x]] <- lowfdr_resList[[x]][order(padj),] %>%
Ranking_fn()
# Rearrange genew with absolute log2FoldChange
lfcList[[x]] <- lowfdr_resList[[x]][order(-abs(log2FoldChange)),] %>%
Ranking_fn()
# Rearrange genes with log2FoldChange (decreasing order)
UPlfcList[[x]] <- lowfdr_resList[[x]][order(-log2FoldChange),] %>%
Ranking_fn()
# Rearrange genes with log2FoldChange (increasing order)
DOWNlfcList[[x]] <- lowfdr_resList[[x]][order(log2FoldChange),] %>%
Ranking_fn()
}
# Explore the ranks
print(c(FDRrankList, lfcList, UPlfcList, DOWNlfcList))## $TPM
## Gene baseMean log2FoldChange lfcSE stat pvalue
## 1: CCN2 3774.26448 3.2928140 0.1897118 17.345356 2.138482e-67
## 2: F2R 2466.06108 1.9013819 0.1194905 15.908801 5.505788e-57
## 3: TM4SF18 1929.69892 2.0213739 0.1403122 14.401312 5.077035e-47
## 4: CDCP1 1520.13638 2.6475329 0.1880305 14.067990 5.974381e-45
## 5: GPR87 559.19270 3.3085620 0.2418261 13.611243 3.433519e-42
## ---
## 482: P2RY13 73.90787 -0.6994790 0.2832717 -2.467164 1.361881e-02
## 483: RBM43 32.83603 -0.9008497 0.3646408 -2.465313 1.368935e-02
## 484: VEGFD 13.48414 -1.0483426 0.4232315 -2.466442 1.364630e-02
## 485: ZFP36 61.12927 0.7549845 0.3054432 2.468805 1.355651e-02
## 486: TSPAN8 78.68030 -0.7002037 0.2843908 -2.461475 1.383672e-02
## padj FDR Input Rank
## 1: 7.420531e-64 < 0.1 TPM 1
## 2: 9.552543e-54 < 0.1 TPM 2
## 3: 5.872437e-44 < 0.1 TPM 3
## 4: 5.182775e-42 < 0.1 TPM 4
## 5: 2.382862e-39 < 0.1 TPM 5
## ---
## 482: 9.794233e-02 < 0.1 TPM 482
## 483: 9.794233e-02 < 0.1 TPM 483
## 484: 9.794233e-02 < 0.1 TPM 484
## 485: 9.794233e-02 < 0.1 TPM 485
## 486: 9.879304e-02 < 0.1 TPM 486
##
## $Counts
## Gene baseMean log2FoldChange lfcSE stat pvalue
## 1: CCN2 3817.8820 3.2844202 0.1915393 17.136222 7.966139e-66
## 2: F2R 2495.7817 1.8984783 0.1186228 16.000704 1.263392e-57
## 3: TM4SF18 1948.8878 2.0182562 0.1399437 14.417068 4.041495e-47
## 4: CDCP1 1537.7433 2.6409502 0.1893300 13.936786 3.786111e-44
## 5: GPR87 565.6044 3.3012898 0.2433261 13.497800 1.611156e-41
## ---
## 494: PCDHA11 137.2504 0.7695862 0.3152421 2.441691 1.461864e-02
## 495: MTND1P23 796.7407 -0.6066005 0.2485948 -2.439717 1.469877e-02
## 496: CHAC2 161.8005 0.6276699 0.2574698 2.436761 1.481945e-02
## 497: MRPL13 3338.7125 0.2996762 0.1230187 2.436026 1.484962e-02
## 498: TIMM8A 370.7887 0.4324878 0.1775077 2.436345 1.483652e-02
## padj FDR Input Rank
## 1: 2.652724e-62 < 0.1 Counts 1
## 2: 2.103547e-54 < 0.1 Counts 2
## 3: 4.486059e-44 < 0.1 Counts 3
## 4: 3.151937e-41 < 0.1 Counts 4
## 5: 1.073030e-38 < 0.1 Counts 5
## ---
## 494: 9.865956e-02 < 0.1 Counts 494
## 495: 9.888266e-02 < 0.1 Counts 495
## 496: 9.929565e-02 < 0.1 Counts 496
## 497: 9.929565e-02 < 0.1 Counts 497
## 498: 9.929565e-02 < 0.1 Counts 498
##
## $TPM
## Gene baseMean log2FoldChange lfcSE stat pvalue
## 1: SLC5A3 175.78052 -5.6388461 0.3735942 -8.890554 6.080480e-19
## 2: EDN1 141.85334 3.4475869 0.3323096 10.072153 7.335473e-24
## 3: MUC12 36.09282 3.3271176 0.4077041 6.359295 2.026818e-10
## 4: GPR87 559.19270 3.3085620 0.2418261 13.611243 3.433519e-42
## 5: CCN2 3774.26448 3.2928140 0.1897118 17.345356 2.138482e-67
## ---
## 482: KPNA4 3914.88812 0.2943474 0.1086781 2.708425 6.760349e-03
## 483: CD2AP 3258.51969 -0.2941260 0.1160457 -2.534561 1.125884e-02
## 484: EIF4EBP2 4482.98608 0.2913268 0.1178709 2.471578 1.345182e-02
## 485: ND1 35661.75864 -0.2884041 0.1035473 -2.785240 5.348811e-03
## 486: TPMT 2885.58887 0.2844551 0.1143035 2.488592 1.282500e-02
## padj FDR Input Rank
## 1: 1.172181e-16 < 0.1 TPM 1
## 2: 2.828232e-21 < 0.1 TPM 2
## 3: 1.562902e-08 < 0.1 TPM 3
## 4: 2.382862e-39 < 0.1 TPM 4
## 5: 7.420531e-64 < 0.1 TPM 5
## ---
## 482: 5.938838e-02 < 0.1 TPM 482
## 483: 8.643403e-02 < 0.1 TPM 483
## 484: 9.765234e-02 < 0.1 TPM 484
## 485: 5.016318e-02 < 0.1 TPM 485
## 486: 9.509136e-02 < 0.1 TPM 486
##
## $Counts
## Gene baseMean log2FoldChange lfcSE stat pvalue
## 1: SLC5A3 178.16943 -5.6507587 0.3735892 -8.902361 5.467110e-19
## 2: EDN1 143.44679 3.4520859 0.3321884 10.088629 6.202983e-24
## 3: GPR87 565.60436 3.3012898 0.2433261 13.497800 1.611156e-41
## 4: CCN2 3817.88197 3.2844202 0.1915393 17.136222 7.966139e-66
## 5: MUC12 21.13784 3.2420717 0.4215420 7.009996 2.383243e-12
## ---
## 494: ND1 36116.54615 -0.2919487 0.1030042 -2.834337 4.592082e-03
## 495: KPNA4 3961.88892 0.2907356 0.1076690 2.700261 6.928505e-03
## 496: EIF4EBP2 4539.93490 0.2878481 0.1178920 2.441629 1.462115e-02
## 497: TPMT 2922.37985 0.2810499 0.1138352 2.468918 1.355223e-02
## 498: IER3IP1 6644.69103 -0.2681817 0.1092941 -2.453759 1.413716e-02
## padj FDR Input Rank
## 1: 1.011415e-16 < 0.1 Counts 1
## 2: 2.295104e-21 < 0.1 Counts 2
## 3: 1.073030e-38 < 0.1 Counts 3
## 4: 2.652724e-62 < 0.1 Counts 4
## 5: 2.088473e-10 < 0.1 Counts 5
## ---
## 494: 4.224208e-02 < 0.1 Counts 494
## 495: 5.840993e-02 < 0.1 Counts 495
## 496: 9.865956e-02 < 0.1 Counts 496
## 497: 9.382315e-02 < 0.1 Counts 497
## 498: 9.666685e-02 < 0.1 Counts 498
##
## $TPM
## Gene baseMean log2FoldChange lfcSE stat pvalue
## 1: EDN1 141.85334 3.447587 0.3323096 10.072153 7.335473e-24
## 2: MUC12 36.09282 3.327118 0.4077041 6.359295 2.026818e-10
## 3: GPR87 559.19270 3.308562 0.2418261 13.611243 3.433519e-42
## 4: CCN2 3774.26448 3.292814 0.1897118 17.345356 2.138482e-67
## 5: SERPINE1 8982.01227 2.857580 0.3273250 8.752297 2.090532e-18
## ---
## 482: TRMT9B 145.09703 -1.725347 0.3138013 -5.481822 4.209682e-08
## 483: WFIKKN2 368.31877 -1.904874 0.2350517 -8.090961 5.919605e-16
## 484: GPR171 242.54169 -2.103719 0.2436843 -8.604146 7.688684e-18
## 485: HLA-DRA 273.70898 -2.141204 0.2510380 -8.510174 1.736718e-17
## 486: SLC5A3 175.78052 -5.638846 0.3735942 -8.890554 6.080480e-19
## padj FDR Input Rank
## 1: 2.828232e-21 < 0.1 TPM 1
## 2: 1.562902e-08 < 0.1 TPM 2
## 3: 2.382862e-39 < 0.1 TPM 3
## 4: 7.420531e-64 < 0.1 TPM 4
## 5: 3.817971e-16 < 0.1 TPM 5
## ---
## 482: 2.001040e-06 < 0.1 TPM 482
## 483: 8.216411e-14 < 0.1 TPM 483
## 484: 1.333987e-15 < 0.1 TPM 484
## 485: 2.739278e-15 < 0.1 TPM 485
## 486: 1.172181e-16 < 0.1 TPM 486
##
## $Counts
## Gene baseMean log2FoldChange lfcSE stat pvalue
## 1: EDN1 143.44679 3.452086 0.3321884 10.088629 6.202983e-24
## 2: GPR87 565.60436 3.301290 0.2433261 13.497800 1.611156e-41
## 3: CCN2 3817.88197 3.284420 0.1915393 17.136222 7.966139e-66
## 4: MUC12 21.13784 3.242072 0.4215420 7.009996 2.383243e-12
## 5: SERPINE1 9084.96822 2.853314 0.3275487 8.733282 2.473876e-18
## ---
## 494: TRMT9B 145.58638 -1.733195 0.3137029 -5.514008 3.507534e-08
## 495: WFIKKN2 373.25309 -1.906352 0.2355697 -8.079008 6.529557e-16
## 496: GPR171 210.73283 -2.093395 0.2444487 -8.557681 1.151604e-17
## 497: HLA-DRA 276.91753 -2.141394 0.2514838 -8.494088 1.994923e-17
## 498: SLC5A3 178.16943 -5.650759 0.3735892 -8.902361 5.467110e-19
## padj FDR Input Rank
## 1: 2.295104e-21 < 0.1 Counts 1
## 2: 1.073030e-38 < 0.1 Counts 2
## 3: 2.652724e-62 < 0.1 Counts 3
## 4: 2.088473e-10 < 0.1 Counts 4
## 5: 4.335793e-16 < 0.1 Counts 5
## ---
## 494: 1.622235e-06 < 0.1 Counts 494
## 495: 8.697370e-14 < 0.1 Counts 495
## 496: 1.917421e-15 < 0.1 Counts 496
## 497: 3.019589e-15 < 0.1 Counts 497
## 498: 1.011415e-16 < 0.1 Counts 498
##
## $TPM
## Gene baseMean log2FoldChange lfcSE stat pvalue
## 1: SLC5A3 175.78052 -5.638846 0.3735942 -8.890554 6.080480e-19
## 2: HLA-DRA 273.70898 -2.141204 0.2510380 -8.510174 1.736718e-17
## 3: GPR171 242.54169 -2.103719 0.2436843 -8.604146 7.688684e-18
## 4: WFIKKN2 368.31877 -1.904874 0.2350517 -8.090961 5.919605e-16
## 5: TRMT9B 145.09703 -1.725347 0.3138013 -5.481822 4.209682e-08
## ---
## 482: SERPINE1 8982.01227 2.857580 0.3273250 8.752297 2.090532e-18
## 483: CCN2 3774.26448 3.292814 0.1897118 17.345356 2.138482e-67
## 484: GPR87 559.19270 3.308562 0.2418261 13.611243 3.433519e-42
## 485: MUC12 36.09282 3.327118 0.4077041 6.359295 2.026818e-10
## 486: EDN1 141.85334 3.447587 0.3323096 10.072153 7.335473e-24
## padj FDR Input Rank
## 1: 1.172181e-16 < 0.1 TPM 1
## 2: 2.739278e-15 < 0.1 TPM 2
## 3: 1.333987e-15 < 0.1 TPM 3
## 4: 8.216411e-14 < 0.1 TPM 4
## 5: 2.001040e-06 < 0.1 TPM 5
## ---
## 482: 3.817971e-16 < 0.1 TPM 482
## 483: 7.420531e-64 < 0.1 TPM 483
## 484: 2.382862e-39 < 0.1 TPM 484
## 485: 1.562902e-08 < 0.1 TPM 485
## 486: 2.828232e-21 < 0.1 TPM 486
##
## $Counts
## Gene baseMean log2FoldChange lfcSE stat pvalue
## 1: SLC5A3 178.16943 -5.650759 0.3735892 -8.902361 5.467110e-19
## 2: HLA-DRA 276.91753 -2.141394 0.2514838 -8.494088 1.994923e-17
## 3: GPR171 210.73283 -2.093395 0.2444487 -8.557681 1.151604e-17
## 4: WFIKKN2 373.25309 -1.906352 0.2355697 -8.079008 6.529557e-16
## 5: TRMT9B 145.58638 -1.733195 0.3137029 -5.514008 3.507534e-08
## ---
## 494: SERPINE1 9084.96822 2.853314 0.3275487 8.733282 2.473876e-18
## 495: MUC12 21.13784 3.242072 0.4215420 7.009996 2.383243e-12
## 496: CCN2 3817.88197 3.284420 0.1915393 17.136222 7.966139e-66
## 497: GPR87 565.60436 3.301290 0.2433261 13.497800 1.611156e-41
## 498: EDN1 143.44679 3.452086 0.3321884 10.088629 6.202983e-24
## padj FDR Input Rank
## 1: 1.011415e-16 < 0.1 Counts 1
## 2: 3.019589e-15 < 0.1 Counts 2
## 3: 1.917421e-15 < 0.1 Counts 3
## 4: 8.697370e-14 < 0.1 Counts 4
## 5: 1.622235e-06 < 0.1 Counts 5
## ---
## 494: 4.335793e-16 < 0.1 Counts 494
## 495: 2.088473e-10 < 0.1 Counts 495
## 496: 2.652724e-62 < 0.1 Counts 496
## 497: 1.073030e-38 < 0.1 Counts 497
## 498: 2.295104e-21 < 0.1 Counts 498Comparing DEG ranks between TPM- and Counts-inputted DE analysis
# Set a function rebuilding DE tables with gene ranks
combineTable_fn <- function(List){
# Select columns and join the data frames by gene
full_join(List[[TvC[1]]][,.(Gene, Input, Rank, baseMean)],
List[[TvC[2]]][,.(Gene, Input, Rank, baseMean)], by="Gene") %>%
# Add columns assining gene expression levels, rank differences, and mean ranks
mutate(logMeanExpression=log(baseMean.x+baseMean.y/2),
RankDiff=Rank.x-Rank.y,
MeanRank=(Rank.x+Rank.y)/2)
}
# Explore outputs of the function
head(combineTable_fn(FDRrankList))## Gene Input.x Rank.x baseMean.x Input.y Rank.y baseMean.y
## 1: CCN2 TPM 1 3774.2645 Counts 1 3817.8820
## 2: F2R TPM 2 2466.0611 Counts 2 2495.7817
## 3: TM4SF18 TPM 3 1929.6989 Counts 3 1948.8878
## 4: CDCP1 TPM 4 1520.1364 Counts 4 1537.7433
## 5: GPR87 TPM 5 559.1927 Counts 5 565.6044
## 6: F2RL2 TPM 6 3010.5527 Counts 6 3047.3796
## logMeanExpression RankDiff MeanRank
## 1: 8.645271 0 1
## 2: 8.219852 0 2
## 3: 7.973894 0 3
## 4: 7.735874 0 4
## 5: 6.735774 0 5
## 6: 8.419413 0 6dim(combineTable_fn(FDRrankList))## [1] 503 10tail(combineTable_fn(FDRrankList))## Gene Input.x Rank.x baseMean.x Input.y Rank.y baseMean.y
## 1: KCTD7 <NA> NA NA Counts 492 248.5733
## 2: PCDHA11 <NA> NA NA Counts 494 137.2504
## 3: MTND1P23 <NA> NA NA Counts 495 796.7407
## 4: CHAC2 <NA> NA NA Counts 496 161.8005
## 5: MRPL13 <NA> NA NA Counts 497 3338.7125
## 6: TIMM8A <NA> NA NA Counts 498 370.7887
## logMeanExpression RankDiff MeanRank
## 1: NA NA NA
## 2: NA NA NA
## 3: NA NA NA
## 4: NA NA NA
## 5: NA NA NA
## 6: NA NA NA# Set a function plotting gene ranks between TPM- (x-axis) and Counts-Inputs (y-axis)
compareRanks_fn <- function(df, rankedby) {
ggplot(df,
aes(x=Rank.x,
y=Rank.y,
color=logMeanExpression)) +
geom_point(alpha=0.5) +
theme_bw() +
xlab("Rank with TPM") +
ylab("Rank with Counts") +
geom_abline(slope=1, color="black", size=0.5) +
ggtitle(paste(rankedby, "Ranking with TPM vs Counts Inputs")) +
scale_color_gradient(low="blue", high="red")
}
# Set a function plotting the rank difference over the gene expression level
RankdiffOverMean_fn <- function(df, rankedby) {
ggplot(df,
aes(x=logMeanExpression,
y=RankDiff,
color=MeanRank)) +
geom_point(alpha=0.5) +
theme_bw() +
ylab("Rank Difference (TPM - Counts)") +
ggtitle(paste("Rank Difference Inputs (TPM - Counts) in", rankedby)) +
geom_hline(yintercept=0, color="black", size=0.5) +
scale_color_gradient(low="blue", high="red")
}Visualizing DEG ranks I: TPM- vs Counts-input
- x-axis: rank with TPM input
- y-axis: rank with Counts input
- Black diagonal lines: rank with TPM = rank with Counts
- Dot color: gene expression level (log-baseMean)
# Ranked by FDR
compareRanks_fn(combineTable_fn(FDRrankList),
"FDR")
# Ranked by absolute fold change
compareRanks_fn(combineTable_fn(lfcList),
"Absolute Log2FoldChange")
# Ranked by magnitude of positive fold change
compareRanks_fn(combineTable_fn(UPlfcList),
"Log2FoldChange (Increased)")
# Ranked by magnitude of negative fold change
compareRanks_fn(combineTable_fn(DOWNlfcList),
"Log2FoldChange (Decreased)")
Results & Discussion 10:
- FDR ranking (top): Genes with high expression levels (red dots) were ranked similarly between two input types which is close to the black diagonal line. Ranking dissimilarity increased in lower ranking ranges (250-500).
- Absolute fold ranking (2nd top): Genes were ranked higher as their expression levels were lower (blue to purple dots). Genes with high expression levels (red dots) showed less difference in the rankings which is close to the black diagonal line.
- Decrease/increase fold ranking (bottom/2nd botton): Highly expressed genes (red dots) showed middle-rankings (150-350). Low-ranking genes tended to be ranked higher with TPM than with Counts as the slope was higher than 1 which is indicated by the black diagonal line.
Visualizing DEG ranks II: Relationship between gene expression level and rank difference
- x-axis: expression level (log-baseMean)
- y-axis: rank difference (rank with TPM - rank with Counts)
- Black horizontal lines: rank with TPM = rank with Counts
- Dot color: average rank
# Ranked by FDR
RankdiffOverMean_fn(combineTable_fn(FDRrankList),
"FDR")
# Ranked by absolute fold change
RankdiffOverMean_fn(combineTable_fn(lfcList),
"Absolute Log2FoldChange")
# Ranked by magnitude of positive fold change
RankdiffOverMean_fn(combineTable_fn(UPlfcList),
"Log2FoldChange (Increased)")
# Ranked by magnitude of negative fold change
RankdiffOverMean_fn(combineTable_fn(DOWNlfcList),
"Log2FoldChange (Decreased)")
Results & Discussion 11:
- Difference in FDR ranking (top): High-ranking genes (blue dots) showed less difference. Genes with high expression levels (higher x-values) showed less difference.
- Difference in absolute/increase fold ranking (2nd top/2nd bottom): Low mean ranks (red dots) seemed to be coupled with higher ranking with TPM (negative y-values) than that with Counts while high mean ranks (purple to blue dots) seemed vice versa or random.
- Difference in decrease fold ranking (bottom): Low-to-middle ranking genes (red-purple dots) tended to have higher ranking with TPM (negative y-values) whereas high ranking genes (blue dots) showed higher ranking with Counts (positive y-values)
Summarizing up/down DEGs with an upset plot
- Calculate the number of genes
# Clean data to generate an upset plot
res <- res %>%
# Filter genes with valid padj
filter(!is.na(padj)) %>%
# Detect genes which are up/down/unchanged change patterns in either TPM and Counts inputs
mutate(Up=ifelse(FDR == FDRv[1] & log2FoldChange > 0, Gene, ""), # What are upregulated genes?
Down=ifelse(FDR == FDRv[1] & log2FoldChange < 0, Gene, ""), # What are downregulated genes?
Unchanged=ifelse(FDR == FDRv[2], Gene, ""), # What are unchanged genes?
TPM_Input=ifelse(Input == "TPM", Gene, ""), # What are the genes from TPM input?
Counts_Input=ifelse(Input == "Counts", Gene, "")) # What are the genes from Counts input?
# Create a list storing groups of interest
upsetInput <- list(Up=res$Up,
Down=res$Down,
Unchanged=res$Unchanged,
TPM_Input=res$TPM,
Counts_Input=res$Counts)
# Create the upset plot
upset(fromList(upsetInput),
sets.x.label="Number of Genes per Group",
order.by="freq") 
Results & Discussion 12:
- No unique genes were detected by count input
Session Info
sessionInfo()## R version 4.0.3 (2020-10-10)
## Platform: x86_64-conda-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.1 LTS
##
## Matrix products: default
## BLAS/LAPACK: /home/mira/miniconda3/envs/r/lib/libopenblasp-r0.3.10.so
##
## locale:
## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
## [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
## [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] stats4 parallel stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] AnnotationDbi_1.52.0 UpSetR_1.4.0
## [3] ggrepel_0.8.2 ggplotify_0.0.5
## [5] gridExtra_2.3 pheatmap_1.0.12
## [7] DESeq2_1.30.0 SummarizedExperiment_1.20.0
## [9] Biobase_2.50.0 MatrixGenerics_1.2.0
## [11] matrixStats_0.57.0 GenomicRanges_1.42.0
## [13] GenomeInfoDb_1.26.0 IRanges_2.24.0
## [15] S4Vectors_0.28.0 tximport_1.18.0
## [17] forcats_0.5.0 stringr_1.4.0
## [19] dplyr_1.0.2 purrr_0.3.4
## [21] readr_1.4.0 tidyr_1.1.2
## [23] tibble_3.0.4 ggplot2_3.3.2
## [25] tidyverse_1.3.0 AnnotationHub_2.22.0
## [27] BiocFileCache_1.14.0 dbplyr_2.0.0
## [29] BiocGenerics_0.36.0 rmarkdown_2.5
## [31] data.table_1.13.2
##
## loaded via a namespace (and not attached):
## [1] colorspace_2.0-0 ellipsis_0.3.1
## [3] XVector_0.30.0 fs_1.5.0
## [5] rstudioapi_0.13 farver_2.0.3
## [7] bit64_4.0.5 interactiveDisplayBase_1.28.0
## [9] fansi_0.4.1 lubridate_1.7.9.2
## [11] xml2_1.3.2 splines_4.0.3
## [13] geneplotter_1.68.0 knitr_1.30
## [15] jsonlite_1.7.1 broom_0.7.2
## [17] annotate_1.68.0 shiny_1.5.0
## [19] BiocManager_1.30.10 compiler_4.0.3
## [21] httr_1.4.2 rvcheck_0.1.8
## [23] backports_1.2.0 assertthat_0.2.1
## [25] Matrix_1.2-18 fastmap_1.0.1
## [27] cli_2.2.0 later_1.1.0.1
## [29] htmltools_0.5.0 tools_4.0.3
## [31] gtable_0.3.0 glue_1.4.2
## [33] GenomeInfoDbData_1.2.4 rappdirs_0.3.1
## [35] Rcpp_1.0.5 cellranger_1.1.0
## [37] vctrs_0.3.5 xfun_0.19
## [39] ps_1.5.0 rvest_0.3.6
## [41] mime_0.9 lifecycle_0.2.0
## [43] XML_3.99-0.3 zlibbioc_1.36.0
## [45] scales_1.1.1 hms_0.5.3
## [47] promises_1.1.1 RColorBrewer_1.1-2
## [49] yaml_2.2.1 curl_4.3
## [51] memoise_1.1.0 stringi_1.5.3
## [53] RSQLite_2.2.1 BiocVersion_3.12.0
## [55] genefilter_1.72.0 BiocParallel_1.24.0
## [57] rlang_0.4.9 pkgconfig_2.0.3
## [59] bitops_1.0-6 evaluate_0.14
## [61] lattice_0.20-41 labeling_0.4.2
## [63] bit_4.0.4 tidyselect_1.1.0
## [65] plyr_1.8.6 magrittr_2.0.1
## [67] R6_2.5.0 generics_0.1.0
## [69] DelayedArray_0.16.0 DBI_1.1.0
## [71] pillar_1.4.7 haven_2.3.1
## [73] withr_2.3.0 survival_3.2-7
## [75] RCurl_1.98-1.2 modelr_0.1.8
## [77] crayon_1.3.4 locfit_1.5-9.4
## [79] grid_4.0.3 readxl_1.3.1
## [81] blob_1.2.1 reprex_0.3.0
## [83] digest_0.6.27 xtable_1.8-4
## [85] httpuv_1.5.4 gridGraphics_0.5-0
## [87] munsell_0.5.0